The Role and Antagonistic Effects of miR-16-5p in the Regulation of ADP-Ribosylation Factor-Like Tumor Suppressor Gene 1 in Lung Cancer Cells

Objective: ADP-ribosylation factor-like tumor suppressor gene 1 is a member of the Ras superfamily of small guanosine triphosphatases that are known to be involved in multiple regulatory pathways in the multistage development of human cancers. Also, ADP-ribosylation factor-like tumor suppressor gene 1 expression levels have been reported to be dramatically lower in both cancer cell lines and tumor tissues compared to controls. Accordingly, defects in the regulation of the ADP-ribosylation factor-like tumor suppressor gene 1 gene seems have key tumor suppressive effects in the formation and development of human cancers including lung cancer. Moreover, microRNAs regulating the expression of ADP-ribosylation factor-like tumor suppressor gene 1 have not been described previously. Accordingly, the present study aimed to reveal the influence of miR-16-5p on the regulation of ADP-ribosylation factor-like tumor suppressor gene 1 gene. Materials and Methods: A549 lung adenocarcinoma cells were used. For the overexpression and silencing experiments of miR-16-5p synthetic microRNA mimics and inhibitors were used, respectively. Gene expression analyses were achieved with the help of quantitative real-time polymerase chain reaction. Results: MiR-16-5p was identified to be predictive target of ADP-ribosylation factor-like tumor suppressor gene 1 and directly targets the expression of ADP-ribosylation factor-like tumor suppressor gene 1 as revealed by the overexpression and silencing experiments. Specifically, it was found that miR-16-5p-overexpressed A549 cells showed a decrease in ADP-ribosylation factor-like tumor suppressor gene 1 gene expression, whereas miR-16-5p-suppressed cells showed an increase in expression. These findings possibly suggest that miR-16-5p is the direct regulatory microRNA that posttranscriptionally regulates the expression of ADP-ribosylation factor-like tumor suppressor gene 1. Conclusion: Collectively, miR-16-5p seems to be a key regulatory molecule involved in the posttranscriptional regulation of the ADP-ribosylation factor-like tumor suppressor gene 1, and it might be responsible for the downregulation of this gene in lung cancer.


Introduction
Lung cancer is the most common malignancy worldwide and the leading cause of cancer-related deaths in men and women.According to 2020 global cancer statistics, 2 206 771 (11.4%) new cases of lung cancer and 1 796 144 (18.0%) lung cancer-related deaths were estimated to occur. 1 Treatment of lung cancer largely depends on the early diagnosis of the disease and its pathological stage.Therefore, the determination of biomarkers that can be used in the early diagnosis of lung cancer is important in the fight against cancer.
ADP-ribosylation factor-like tumor suppressor gene 1 is a member of the ARF family, the Ras superfamily of small guanosine triphosphatases known to be involved in numerous regulatory pathways in the multistage developmental process of human cancers. 2 Structural and functional disorders caused by the ARLTS1 gene in cancer are of 3 types: genomic loss, gene polymorphisms, and epigenetic changes.The main reason for downregulation of ARLTS1 appears to be promoter methylation. 3In addition, ARLTS1 expression levels have been reported to be dramatically lower in both cancer cell lines and tumor tissues compared to controls.Accordingly, defects in the regulation of the ARLTS1 seem to be a key factor in the formation and development of human cancers. 2,4e Role and Antagonistic Effects of miR-16-5p in the Regulation of ADP-Ribosylation Factor-Like Tumor Suppressor Gene 1 in Lung Cancer Cells Previously, it was assumed that only oncogenes and tumor suppressor genes were involved in the molecular basis of cancer.However, with the discovery of noncoding RNA (ncRNA) molecules, which play significant regulatory roles in regulating gene expression, this assumption has largely lost its validity. 5The studies conducted in the following years allowed the identification of numerous classes of noncoding RNAs, in particular long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), which are involved in the regulation of gene expression and exhibit oncogenic/tumor suppressive properties. 6In particular, miRNAs have attracted great interest among noncoding RNAs because they are more stable and more easily targeted therapeutically. 7icroRNAs are small noncoding RNAs about 18-22 nucleotides long. 8MicroRN induce posttranscriptional repression of their target genes by showing partial complementarity to the UTR (untranslated region) of their target mRNAs.The production of mature RNA sequences in the cell takes place through a series of processing events. 5Recent studies have shown that miRNAs mediate various cellular processes, such as cell cycle control and regulation of cell death mechanisms, through various targets. 6R-16, which has significant regulatory roles in the development and progression of human cancers, is localized in the intronic region of the Deleted in Lymphocytic Leukemia 2 (DLEU2) lncRNA gene mapped in the 13q14.2chromosomal region. 9The miR-16 has been shown to negatively regulate and antagonize B cell lymphoma 2 (Bcl-2) to induce apoptosis. 10It has been shown that miR-16 regulates the expression of cyclin D1 (CCND1), cyclin D3 (CCND3), and cyclin E1 (CCNE1) to induce G0/G1 cell cycle arrest. 9Accumulating evidence also suggests that the expression level of miR-16 is low in the tumor tissues and lung cancer cells of non-small-cell lung cancer patients. 11,12reover, miRNAs regulating ARLTS1 expression have not been described previously.Accordingly, the present study aimed to uncover the impact of miR-16-5p on ARLTS1 regulation.To uncover the effects of miR-16-5p on ARLTS1 regulation, overe xpres sion/ silen cing experiments were performed with synthetic miRNA mimics/inhibitors in A549 lung cancer cells.

miR-16 Mimic and Inhibitor Transfections
Mimic and inhibitor experiments were performed to overexpress and suppress miR-16-5p in A549 cells.miR-16-5p mimics, inhibitors, and a negative control, which have no detectable target, were commercially obtained (Qiagen, Germany).For the transfection experiments, cells were seeded into 12-well plates at a concentration of 5 × 10 4 cells/well.For the transfections, HiPerFect Transfection Reagent (Qiagen, Hilden, Germany) were used.The following transfection mixture was prepared: 100 µL DMEM, 4 µL HiPerFect Transfection Reagent (Qiagen, Germany), and 0.5 µL mimic or 4 µL inhibitor.The prepared mixture was incubated at room temperature for 30 minutes and delivered to cells dropwise and 10% fetal calf serum was added after about 4 hours.Following 72 hours post transfection, cells were removed for total RNA isolation.

RNA Extractions and cDNA Synthesis
Following transfections, cells were collected and total RNA was extracted with the help of the miRNeasy Mini Kit (Qiagen, Germany).Cells were first lysed using Qiazol reagent, and recommendations of the manufacturer were followed.After the isolations, the RNA concentrations were measured using a NanoDrop 2000.RNA concentrations were approximately 100-1000 ng/µL and further adjusted to 500 ng/µL.For the synthesis of complementary DNA (cDNA), the miScript II RT kit (QIAGEN, Germany) was used.HiFlex buffer was used for cDNA synthesis of long RNAs.For the synthesis, 500 ng/µL RNA, 4 µL HiFlex buffer, 2 µL Nuclei Mix, 2 µL Reverse Transcriptase, and nuclease-free water up to 20 µL were used.The prepared mixture was incubated at 37°C for 60 minutes and 95°C for 5 minutes, and after the reaction, it was immediately placed on ice and stored at -80°C.

Quantitative Expression nalysis by Real-Time Polymerase Chain Reaction
MiScript SYBR Green PCR Kit (Qiagen, Germany) and miR-16 primer assay kit were used to determine miR-16 expression levels.
RNU6 was used as the reference gene to determine the expression level of miR-16.The reaction mixture prepared was subjected to thermal conditions at 94 °C for 15 minutes, 40 cycles at 94 °C for 15 seconds, at 55 °C for 30 seconds, and at 70 °C for 30 seconds.For the determination of ARLTS1 gene expression levels, gene-specific primers (F primer: 5'-CC CATCT CGCTC TTGGG TCAT-3'; R primer: 5'-CC ACTGC TGAAT CCTCT GTGTT -3') and RealQ Plus 2x Master Mix (Ampliqon PCR Enzymes & Reagents, Denmark) were used.GAPDH (F primer: 5'-GA TCATC AGCAA TGCCT CCT-3 '; R primer: 5'-TG TGGTC ATGAG TCCTT CCA-3 ') was used as a reference to determine the expression level of ARLTS1.The prepared reaction mixtures were incubated at following thermal conditions; 95 °C for 15 minutes, 40 cycles at 95 °C for 15 seconds, at 60 °C for 30 seconds, at 72 °C for 30 seconds.Cycle threshold values were calculated at the appropriate threshold value following all reactions.All reactions were repeated 3 times.

Statistical Analyses
All statistical evaluations were performed using GraphPad Prism, Version 8.For the calculation of gene expression levels of miR-16 and ARLTS1, 2 -ΔCt (ΔCt = Ct Target Gene -Ct Reference Gene) formula was used.Student' s t-test was used for comparisons between variables.For all results, those with P <.05 were considered significant.

Determination of miRNA Target by Using miRNA-Target Prediction Algorithms
To determine the miRNAs posttranscriptionally targeting ARLTS1 gene TargetScan 13 miRNAtarget prediction algorithm was used and miR-16-5p was selected.The most important criterion in determining the relevant miRNA was that the selected miRNA was not previously reported to be associated with ARLTS1 in the literature.In the TargetScan database, it was determined that ARLTS1 showed 7mer-m8 complementarity with miR-16-5p between positions 755-761 in the 3'-UTR region.As a result, it was

Main Points
• In this study, the interaction between miR-16 and the ARLTS1 gene was determined.
• miR-16-5p seems to be a key regulatory molecule in the regulation of ARLTS1 gene.

Overexpression and Silencing of miRNA-16-5p
To further explore the interaction between ARLTS1 and miRNA-16-5p, overexpression and silencing experiments were performed with synthetic miRNA mimics and inhibitors, respectively, in A549 lung cancer cell lines.After miR-16-5p mimic and inhibitor transfections, miR-16-5p level was determined by quantitative polymerase chain reaction.As a result, it was found that the expression level of miR-16-5p increased approximately 10 times in the mimic transfected group compared to the negative control (P <.0001) and decreased approximately 1.6 times in the inhibitor transfected group compared to the negative control (P = .0032)(Figure 2).These results were found to be statistically significant.

ARLTS1 Expression Levels Following Mimic/ Inhibitor Transfections
To examine the impact of overexpression and silencing of miRNA-16-5p on ARLTS1, expression levels of ARLTS1 were determined.As a result, ARLTS1 expression level was found to be significantly decreased by approximately 1.7 times in miR-16-5p overexpressing cells (P = .0107).In contrast, the expression level of ARLTS1 increased approximately 2-fold in cells in which miR-16-5p inhibitor was applied and miR-16-5p expression levels were suppressed (P = .0002)(Figure 3).

Discussion
In this study, first, the interaction between miR-16 and the ARLTS1 was determined by bioinformatics tools, and then overexpression and suppression experiments were performed in A549 lung cancer cells after miR-16 mimic and inhibitor transfections.Notably, it was found that the expression level of the ARLTS1 decreased in miR-1 6-5p-overe xpres sed A549 cells and increased in miR-16-5p-suppressed cells, strongly suggesting that miR-16-5p is chief regulatory miRNA that posttranscriptionally fine-tune the expression of ARLTS1.Overall, we report here for the first time an as yet unidentified novel mechanism for posttranscriptional regulation of ARLTS1.miR-16-5p functions as an antagonistic molecule to silence the expression of ARLTS1, indicating that it might be used to modulate expression and functions of ARLTS1.
Although limited, a few studies suggest that ARLTS1 expression is downregulated in various types of human malignant disease, including prostate cancer, 14 lung carcinomas, B cell chronic lymphocytic leukemia, ovarian carcinomas, and ovarian and breast cancer cell lines. 4The limited number of studies demonstrating ARLTS1 expression makes it difficult to comment further on this issue.In addition, findings of the significant number of studies strongly indicate that genomic variations of ARLTS1 is associated with the increased risk of breast cancer, 15,16 multiple melanomas, 17 colorectal cancer, 18 prostate cancer, 19 and ovarian cancer. 20In contrast to these studies, an accumulating body of evidence also suggests that there is no association between ARLTS1 variants and chronic lymphocytic leukemia, 21 colorectal cancer, 22 basal cell carcinoma of the skin, 23 sporadic breast cancer, prostate cancer, melanoma, thyroid papillary cancer, or laryngeal cancer. 24rthermore, Yendamuri et al showed that overexpression of the ARLTS1 in lung cancer cells reduced cell proliferation, stimulated apoptosis, and reduced the tumorigenic effect in vivo. 25Particularly, in 9 lung cancer cell lines and 27 pairs of normal and lung cancer tissues, ARLTS1 has been shown to be downregulated in all lung cancer cell lines and in approximately 37% of lung tumors compared to normal tissues. 25ADP-ribosylation factor-like tumor suppressor gene 1 was also shown to be associated with the inhibition of cell proliferation in ovarian and breast cancers. 4These data show that ARLTS1 plays a tumor suppressor role in the formation and progression of common malignancies.However, there are no studies on miRNAs targeting the ARLTS1.On the other hand, miR-16-5p has been shown to be poorly expressed in many cancer types.[28]

Conclusion
In conclusion, the present findings identify miR-16-5p as a key regulatory molecule involved in the posttranscriptional regulation of the ARLTS1.The present study also suggests that deregulation of miR-16-5p might be responsible for the downregulation of ARLTS1 in lung cancer.However, the current study has several limitations.Future more comprehensive studies are needed to fully decipher the functions and fate of the ARLTS1 in human cancers.Particularly, gain-of-function and/or loss-of-function experiments of ARLTS1 together with the mimic/inhibitor manipulations will be more valuable to better understand the miR-16-5p/ARLTS1 axis in lung cancer.Also, the use of additional lung cancer cell lines and normal lung epithelial cells will be more valuable to fully comprehend more accurate results.Additionally, a luciferase reporter assay will be more valuable to evaluate the direct interaction between miR-16-5p and the ARLTS1 gene.Also, further experiments with tumor xenograft models and/  or genetically or chemically engineered animal models of lung cancer will be more valuable to understand better about the miR-16-5p-mediated regulation of ARLTS1.
Ethics Committee Approval: N/A.
Informed Consent: N/A.

Declaration of Interests:
The authors have no conflicts of interest to declare.

Funding:
The authors declared that this study has received no financial support.